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Clinical Implication of Hot Spot BRAF Mutation, V599E, in Papillary Thyroid Cancers

Department of Molecular Medicine (H.N., T.I.R., A.O., S.Y.), Tissue and Histopathology Section; Division of Scientific Data Registry (M.N.), International Health and Radiation Research (V.A.S., S.Y.), Atomic Bomb Disease Institute; Departments of Pathology (T.H.) and Division of Endocrine Surgery (S.M.); and Department of Surgery (N.H., T.K.), Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 852-8523, Japan

Address all correspondence and requests for reprints to: Prof. Hiroyuki Namba, M.D., Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: namba{at}net.nagasaki-u.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activating mutations in the BRAF kinase gene have recently been reported in human cancers. The aim of the present study was to determine the frequency of BRAF mutations in thyroid cancer and their correlation with clinicopathological parameters. We analyzed exons 11 and 15 of BRAF gene in six human thyroid cancer cell lines and 207 paraffin-embedded thyroid tumor tissues. A missense mutation was found at T1796A (V599E) in exon 15 in four of the six cell lines and 51 of 207 thyroid tumors (24.6%; 0 of 20 follicular adenoma, 0 of 11 follicular carcinoma, 49 of 170 papillary carcinomas, and 2 of 6 undifferentiated carcinomas). Activation of MAPK kinase-MAPK pathway was observed in cell lines harboring BRAF mutation. BRAF mutation-associated enhanced cell growth was suppressed by MAPK kinase inhibitor, U0126. Examination of 126 patients with papillary thyroid cancer showed that BRAF mutation correlated significantly with distant metastasis (P = 0.033) and clinical stage (P = 0.049). Our results indicate that activating mutation of BRAF gene could be a potentially useful marker of prognosis of patients with advanced thyroid cancers.

	Introduction

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THE BIOLOGICAL BEHAVIOR of thyroid cancer varies widely from indolent microcarcinoma, growing slowly with little or no invasion, to invasive cancer that metastasizes and can potentially cause death (1, 2). Previous studies using multifactorial analysis of clinical risk factors in thyroid cancer showed that metastasis, age, completeness of resection, invasion, and tumor size are useful prognostic factors of differentiated thyroid cancer (3). Despite vigorous molecular analysis performed over the past 10 yr, limited prognostic biomarkers are currently available for human thyroid cancers. Ret/PTCs, Ret protooncogene rearrangements, are specifically found in papillary cancers but do not correlate with the grade of malignancy (4). In contrast, our previous studies and those of other investigators have shown that mutations of p53 gene are exclusively found in undifferentiated thyroid cancers (5). Analysis of p53 gene is, therefore, a useful tool to detect undifferentiated thyroid cancers. Considered collectively, there is a desire to identify more reliable prognostic markers such as oncogenes, activated signaling pathways, and other basic mechanisms that are specifically relevant to thyroid cancers.

The Ras/Raf/MAPK kinase (MEK)/MAPK pathway is a classic signal pathway known to mediate cellular proliferation in various cell types. Activating mutations of ras gene are identified in approximately 30% of human thyroid tumors, suggesting that the kinase pathway is involved in thyroid tumorigenesis (6, 7). Recently, activating mutations in the BRAF kinase gene were described in a broad range of other human malignancies (8). The frequency of BRAF mutations varies widely in human cancers from more than 80% in melanomas and nevi (9, 10), to as little as 0–18% in other tumors, such as 1–3% in lung cancers and 5% in colorectal cancers (11, 12, 13). Herein, we investigated the frequency of BRAF mutations and the relationship between the mutation and clinical stage of human thyroid cancers. We detected BRAF mutation, V599E, in four of six human thyroid cancer cell lines and in 51 of 207 thyroid tumor tissues. The correlation analysis using various clinicopathological parameters revealed that BRAF mutation was significantly associated with advanced thyroid cancers.

	Materials and Methods

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Cell culture and materials

Four human thyroid cancer cell lines, ARO, FRO, NPA, and WRO, were kindly provided by Dr. G. Juillard (University of California–Los Angeles, Los Angeles, CA). Another papillary thyroid cancer cell line, TPC-1, and anaplastic carcinoma cell line, KTC-1, were kindly provided by Dr. Sato (Cancer Institute, Kanazawa University, Japan) and Dr. Kurebayashi (Kawasaki Medical School, Kawasaki, Japan) (14), respectively. All cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and grown at 37 C in 5% CO₂-95% air environment.

Thyroid tumor tissues were selected from 207 paraffin blocks (20 follicular adenomas, 170 papillary carcinomas, 11 follicular carcinomas, and six undifferentiated carcinomas) filed at the Department of Pathology, Nagasaki University School of Medicine (Nagasaki, Japan) and Ishigaki Thyroid Clinic (Hamamatsu, Japan). All thyroid tumors were independently reclassified by two experienced pathologists based on the histopathological typing of the World Health Organization as papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, or follicular adenoma (15).

Correlations between BRAF mutation and various clinicopathological parameters were clinically and retrospectively analyzed in 126 patients who consented to the study. Clinical staging of thyroid cancer cases was classified according to the Tumor Node Metastasis (TNM) classification of the International Union Against Cancer (UICC). The study protocol was approved by the Human Ethics Review Committees of Nagasaki University School of Medicine.

Immunoblot analysis

All cells were seeded at a density of 1 x 10⁶ cells in 10-cm dishes. The cells were incubated in RPMI 1640 with 10% FBS for 24 h, and then the medium was changed to RPMI 1640 with 2% FBS. After 24 h, the cells were harvested with RIPA buffer. In the next step, 40 µg of whole cell lysates were separated by electrophoresis in 10% SDS-PAGE, and then blotted onto nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). To quantitate the levels of MEK, phospho-MEK, MAPK, and phosphor-MAPK, the blots were incubated for 60 min with the respective antibody against human MEK, phospho-MEK, MAPK and phosphor-MAPK (Cell Signaling Technology, Beverly, MA). The antigen-antibody complexes were visualized with horseradish peroxidase-conjugated antirabbit IgG antibody and the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Cell growth assays

The kinetics of cell growth were examined using a cytometer as follows. Cells were seeded at a density of 0.1 or 0.5 x 10⁵ cells per well in 12-well culture plates. They were counted at d 2, 3, 4, and 5. The experiments were performed at least three times. Cells were cultured with or without 5 µM U0126 (Cell Signaling Technology), or 0.1% dimethyl sulfoxide and counted at 24 h after treatment.

DNA isolation and sequencing

Genomic DNA was extracted from cell lines using the Wizard Genomic Purification Kit (Promega, Madison, WI) and amplified for analysis of mutations in exons 11 and 15 of BRAF gene (8) and the regions containing codons 12, 13, 59, and 61 of H, K, and N-ras genes by PCR using specific primers (11). DNA from 207 paraffin-embedded thyroid tumor specimens was prepared from five 10-µm-thick sections after microdissection, resulting in selection of more than 90% tumor cells. Genomic DNA was isolated using DXPAT (Takara Co., Kyoto, Japan), and BRAF exons 11 and 15 were amplified by PCR. The following intron-based PCR primers were designed to amplify the exons 11 and 15: BRAF exon 11, forward-TCCCTCTCAGGCATAAGGTAA, reverse-CGAACAGTGAATATTTCCTTTGAT; BRAF exon 15, forward-TCATAATGCTTGCTCTGATAGGA, reverse-GGCCAAAAATTTAATCAGTGGA. PCRs were performed using standard PCR conditions (95 C x 5 min; 94 C x 30 sec, 58 C x 30 sec, 72 C x 30 sec, for 40 cycles; then 70 C x 10 min). The amplified products were purified by MinElute PCR Purification Kit (Qiagen, Chatsworth, CA) and sequenced on an ABI PRISM 3100 automated capillary DNA Sequencer using the BigDye terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA).

Statistical analysis

Data (shown in Table 3) were analyzed using the Mann-Whitney U test or ² for independence test. A P value <0.05 denoted the presence of a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

To detect mutations in the BRAF gene in human thyroid cancer cells, we first performed sequence analysis of BRAF exons 11 and 15 using genomic DNA extracted from six human thyroid cancer cell lines. We found the missense mutation T1796A (V599E) in four of six thyroid cancer cell lines. Among the four cell lines harboring the mutation, homologous mutation was detected in two cell lines, FRO and NPA, and heterologous mutation in the other two cell lines, ARO and KTC-1 (Fig. 1A).

View larger version (40K): [in this window] [in a new window]   FIG. 1. BRAF mutation and its cell proliferative effect in thyroid cancer cell lines. A, Sequencing of BRAF exon 15 in thyroid cancer cell lines. A representative sequence of wild-type BRAF (WRO), heterologous T1796A (V599E) mutation of BRAF (ARO) and homologous mutation (NPA). B, Protein expression levels of p-MEK, MEK, p-MAPK, and MAPK in five thyroid cancer cell lines. C, Cell growth assays performed using MTT assay. Red line, ARO cell; green line, FRO cell; blue line, TPC-1 cell; black line, WRO cell. D, Effects of 24-h treatment with 5 µM U0126, a MEK inhibitor, on thyroid cancer cell lines.

Next, we examined whether activated RAS was involved in the activation of MEK-MAPK pathway. Sequence analysis confirmed no activating mutations of H, K, and N-ras genes in all cell lines used in this study. Table 1 summarizes the results of BRAF, ras, and RET genes mutation analyses in the six human thyroid cancer cell lines. Furthermore, to investigate whether the BRAF mutation affects cell proliferation, cell growth assays were performed. Cell lines with BRAF mutation showed more rapid cell growth than the WRO cell line, which does not harbor BRAF mutation or RET/PTC rearrangement (Fig. 1C). Twenty-four-hour treatment of cells with 5 µM U0126, a MEK1/2 inhibitor, showed significant suppression of cell growth in BRAF mutation cell lines, ARO and FRO, but not in non-BRAF mutation cell lines, TPC-1 and WRO (Fig. 1D). These results suggest that BRAF mutation promotes cell growth directly through the MEK-MAPK pathway in these thyroid cancer cell lines.

We studied BRAF exons 11 and 15 in 207 paraffin-embedded thyroid tumors, including 20 follicular adenomas, 11 follicular carcinomas, 170 papillary carcinomas, and six undifferentiated carcinomas, of 165 female and 42 male patients aged from 12–85 yr (mean, 52 yr) at the time of operation. Of 207 thyroid tumors studied, there were 51 cases (24.6%) with BRAF mutation. Although we examined both BRAF exons 11 and 15, the mutations were limited to the T1796A (V599E) in exon 15. No mutations of BRAF were detected in the normal thyroid tissues surrounding malignant tissue in the six examined BRAF mutation-positive thyroid cancers, suggesting that the mutations were somatically acquired. Further analysis according to tumor type showed that of the 51 thyroid tumors with BRAF mutation (Table 2), none was follicular adenoma or follicular carcinoma, 49 were papillary carcinomas, and two were undifferentiated carcinomas.

We examined the correlation between BRAF mutation and various clinicopathological parameters in 126 patients with papillary thyroid cancer (Table 3). There was no significant correlation between BRAF mutation and sex, age, nodal metastasis, or extrathyroidal invasion at a median postoperative follow-up period of 6 yr. However, there was a significant correlation between BRAF mutation and clinical stage (P = 0.049; Mann-Whitney U test) and distant metastasis to lung or bone (P = 0.033; ² for independence test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here, mutations of BRAF exons 11 and 15 were examined in six human thyroid cancer cell lines and 207 thyroid tumor tissues. These two exons were specifically selected because all previously reported BRAF mutations were identified within these two exons (9). Unlike with other types of cancer, only T1796A (V599E) missense mutation was observed in four of six thyroid cancer cell lines and 51 of 207 thyroid tumors. Of 51 thyroid tumors with BRAF mutation, there were 49 papillary thyroid carcinomas and two undifferentiated carcinomas. Because undifferentiated carcinoma arises from differentiated carcinoma, including papillary carcinoma, BRAF mutation seemed to be papillary phenotype-specific in thyroid tumors. In contrast, BRAF mutation was observed in different types of melanocytic nevi and melanomas at high rates, which indicates that the mutation is a critical initiation step in melanocytic neoplasia (10). Thus, our results and those of previous studies suggest that BRAF mutation plays a different role in the development of tumors in a tissue-type specific manner.

In this study, we found the significant correlation between with BRAF mutation and clinical stage. Thyroid cancers with BRAF mutation were characterized as advanced cancers with metastasis. Consistent with our finding, Webb et al. (17) demonstrated experimentally that the Raf/MEK/MAPK pathway mediates metastasis as well as tumor growth. Our results suggest that BRAF mutation could be a useful marker of poor prognosis of patients with thyroid cancer.

Because activating ras mutations exist in about 30% of thyroid tumors (18), we examined the ras gene mutations in BRAF mutation-positive tumors. No H, N, and K-ras mutations were detected in these tumors (data not shown). Mutations of ras genes have been described in both follicular adenomas and follicular carcinomas, suggesting that ras activation is an early step in thyroid tumorigenesis (19). In contrast, BRAF mutation is mainly associated with the papillary phenotype of differentiated thyroid cancers and cancers of clinically advanced stage. These results suggest that activation of RAS and that of BRAF play different roles in thyroid tumorigenesis, although both molecules activate MAPK. Sirakawa et al. (20) have demonstrated that activation of RAS induces apoptosis of thyroid cells. Activated RAS may affect not only MAPK but also other pathway(s) predisposed to apoptosis. Thus, it seems that the comprehensive effects of constitutive activation of MAPK pathway and other intracellular signals, which are simultaneously activated by the mutation of component genes forming the Ras/Raf/MEK/MAPK pathway, determine the histopathological phenotype and/or aggressiveness of human thyroid tumors.

Because MAPK is thought to be essential for cellular growth in various cancers, this pathway is a target for pharmacological intervention in proliferative diseases (21). In particular, inhibition of MEK represents a suitable target for therapy because of its substrate specificity. In this study, U0126, which inhibits phosphorylation of MEK1/2, suppressed cell growth in BRAF mutation-positive cell lines. Small molecule inhibitors of MEK1/2 have already been developed, and one of them induces potent growth inhibition of colorectal tumors in vivo (22). Such inhibitors may be used orally as noncytotoxic agents for clinical management of patients with thyroid advanced cancers in the near future.

In conclusion, our study provided clinical evidence that BRAF mutation, V599E, correlates with advanced pathological stage in papillary thyroid cancers. The search for BRAF mutation seems to be useful and valuable for evaluation of prognosis of patients with papillary thyroid cancer.

	Acknowledgments

 
We thank Drs. Jitsuhiro Ishigaki and Katsu Ishigaki (Ishigaki Thyroid Clinic, Hamamatsu, Japan) for providing paraffin-embedded tissue blocks, and Ms. Tomoko Kamiya for the excellent technical assistance. We also thank Dr. Kurebayashi for kindly providing KTC-1 cell line.

	Footnotes

 
This work was supported by Grants-in-Aid for Scientific Research 13671158, 14380256, and 12576020 from The Ministry of Education, Culture, Sports, Science and Technology.

Abbreviations: FBS, Fetal bovine serum; MEK, MAPK kinase.

Received February 21, 2003.

Accepted May 18, 2003.

	References

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